Endocytic invagination and vesicle scission - interplay between dynamin homologues and amphiphysins in budding yeast

胞吞内陷和囊泡分裂——酵母中动力蛋白同源物和两性蛋白之间的相互作用。它包括少量的细胞外膜（质膜）被拉入细胞内，直到其中一些膜被挤压形成一个叫做囊泡的小球体。这个囊泡将包含来自细胞外的液体，在它的膜内，它将包含表面的蛋白质。细胞想要从表面移除这些蛋白质，可能是因为它们受损了，或者因为它们可以结合或响应细胞不再想要或需要响应的外部信号。胞吞作用是细胞控制其表面物质的一种非常重要的方式。一些病原体或毒素可以与细胞表面的蛋白质结合并引发内吞作用。通过这种方式，这些不合适的物质可以进入细胞。在一些神经系统疾病如阿尔茨海默病中，内吞过程的缺陷也被早期发现。Ayscough实验室的研究使用一种简单的单细胞生物酿酒酵母（面包酵母）作为模型系统。在这种细胞类型和哺乳动物等更复杂的生物细胞中，许多过程都以同样的方式发生。我们对两类蛋白质的作用特别感兴趣——动力蛋白和两栖蛋白。这些蛋白质被认为参与胞吞作用，但它们起作用的确切步骤一直难以阐明。这样做的原因是，许多有关哺乳动物蛋白质的研究都是用纯化的蛋白质进行的。然后将这些数据转化为生理背景并不总是那么容易。操纵各种哺乳动物的系统并不总是那么简单，有些实验可能需要几个月的时间来完成。酵母提供了一个更简单的情况来研究，我们可以在整个生物体的背景下研究事物。我们使用荧光标记蛋白质的成像来研究感兴趣的蛋白质如何在细胞中移动。我们可以确定蛋白质何时定位到内吞作用的位置以及它们在那里停留的时间。这种成像需要非常敏感，因为内吞部位只有微米大小的几分之一。此外，实际的膜内陷和断裂事件发生在秒的时间尺度上。使用酵母，我们可以很容易地研究改变动力蛋白或两角蛋白中单个氨基酸的影响。除了使用活细胞成像，我们还使用电子显微镜。这允许对内吞作用的关键阶段进行更详细的分析。特别是，我们可以确定基因缺失或突变对膜曲率和囊泡断裂过程的影响，比光学显微镜更确定。我们的方法将在分子水平上对蛋白质的功能提供新的见解。反过来，这将为在健康和患病细胞类型的背景下研究这些蛋白质的其他系统提供信息。

'Endocytic Invagination and Vesicle Scission - interplay between dynamin homologues and amphiphysins in yeast' Ayscough and Goldberg Endocytosis is an essential process in most eukaryotic cells. It involves a small amount of the outer (plasma) membrane of the cell being pulled inwards into the cell until some of this membrane pinches off to form a little sphere called a vesicle. This vesicle will contain fluid from outside the cell and within its membrane it will contain proteins that were on the surface. A cell may want to remove these proteins from the surface because they are damaged, or because they can bind or respond to signals from outside that the cell no longer wants, or needs to respond to. Endocytosis is a very important way for a cell to control what is on its surface. Some pathogens or toxins can bind to proteins on the cell surface and trigger endocytosis. In this way these inappropriate substances can gain entry to the cell. Defects in the endocytic process have also been detected early in some neurological disorders such as Alzheimers. Research in the Ayscough laboratory uses a simple one-celled organism Saccharomyces cerevisiae (bakers yeast) as a model system. Many processes are known to happen in the same way in this cell-type and in cells of more complex organisms such as mammals. We are particularly interested in the role of two classes of proteins - the dynamins and the amphiphysins. These proteins are proposed to be involved in endocytosis but the exact step at which they function has been difficult to elucidate. The reason for this, is that much work on the relevant mammalian proteins has been performed with purified proteins. It is not always easy to then translate this data into a physiological context. Manipulating the various mammalian systems has not always been straightforward and some experiments can take months to perform. The yeast provides a more simple situation to investigate, and we can study things within the context of the whole organism. We use imaging of fluorescently tagged proteins to investigate how the proteins of interest move in the cell. We can determine when the proteins localise to sites of endocytosis and how long they stay there. This imaging needs to be very sensitive as the endocytic sites are only fractions of a micron in size. Furthermore, the actual membrane invagination and scission events occur on a seconds timescale. Using yeast we can readily investigate the effect of changing just single amino acids within the dynamin or amphiphysin proteins. As well as using live cell imaging we use electron microscopy. This allows a much more detailed analysis of the key stages of endocytosis. In particular, we can determine the impact of gene deletions or mutations on the proceses of membrane curvature and vesicle scission with much more certainty than is possible with light microscopy. Our approach will give new insights into the functioning of the proteins at the molecular level. In turn this will inform approaches in other systems studying these proteins in the context of both healthy and diseased cell types.